Recent data from our lab and others have demonstrated that targeted alpha-synuclein overexpression in the substantia nigra pars compacta leads to Parkinson-like neurodegeneration. Yet, the function of alpha synuclein is still unknown. The present proposal attempts to define the function of alpha synuclein in both normal brain function and in the pathogenesis of Parkinson disease (PD). Alpha synuclein is likely to be part of a multiprotein complex, and understanding the interaction between alpha synuclein and its protein binding partners should help in understanding the mechanism underlying pathogenesis in PD, as well as its function in the normal brain. It might also help explain the selective vulnerability of certain neuronal populations to alpha synuclein. We propose to use recombinant Adeno-Associated Virus (rAAV) as a gene delivery vector to generate regionally specific somatic transgenics of the nigrostriatal tract. The following 3 aims are proposed. The first aim is to examine the effect of down regulating or overexpressing genes that are known to interact with alpha synuclein. Phospholipase D2 (PLD2) and/or G coupled Receptor protein Kinase 2 (GRK2) will be overexpressed or knocked down in combination with alpha-synuclein to determine whether the known interactions between these proteins and alpha synuclein are involved in the pathogenesis of PD. We will follow the progression of neurodegeneration caused by these combinations by looking at the number of TH- positive neurons and behavioral deficits. Aim 2 is to identify additional proteins that interact directly with alpha synuclein. Tagged rat and human alpha synuclein will be used as a bait to affinity-immunoprecipitate a synuclein-protein complexes in vivo in the dopaminergic MN9D cell line. The affinity purified complexes will be analyzed by mass spectrometry methods and antibody to known interaction partners to identify the multiprotein interactions for alpha synuclein. Aim 3 is to determine whether the toxicity of dopamine related oxidative stress is a factor in the selective vulnerability of certain dopaminergic neurons to Parkinson-like neurodegeneration induced by alpha synuclein. AAV will be used to deliver the alpha synuclein gene alone, or in combination with genes that increase or decrease oxidative stress in substantia nigra. These include the tyrosine hydroxylase (TH) and GTP cyclohydrolase genes to increase nigrostriatal dopamine production by overexpression of the precursor L-dopa, and SOD1 and catalase to reduce oxidative stress. As in aim 1 we will follow the progression of neurodegeneration caused by these combinations.